The present invention is directed to the use of a refined detoxified endotoxin (RDE) product as a potent immunostimulator and also as an adjuvant. The RDE used in the present invention is characterized as having no detectable 2-keto-3-deoxyoctanoate, between about 350 and 475 nmoles/mg of phosphorus and between about 1700 and 2000 nmoles/mg of fatty acids. Reference is made to U.S. Pat. No. 4,436,727 which describes the RDE product in detail including the method of preparation and which is incorporated herein.
Endotoxic extracts obtained from Enterobacteriaciae including parent organisms and mutants are known. These extracts have been used for immunotherapy of various immunogenic tumors [see Peptides as Requirement for Immunotherapy of the Guinea-Pig Line-10 Tumor with Endotoxins; Ribi, et al. Cancer Immunol. Immunother., Vol. 7, pgs. 43-58 (1979) incorporated herein by reference]. However, the endotoxin extracts are known to be highly toxic and, therefore, of limited use in the treatment of cancerous tumors. Efforts have been made to "detoxify" the endotoxins while retaining its tumor regressive capacity. As shown, in Ribi, et al., supra, chemical procedures known to "detoxify" endotoxins while retaining adjuvanticity, such as succinylation and phthalylation resulted in both loss of endotoxicity and tumor regressive potency. Therefore, prior art attempts to obtain a refined detoxified endotoxin product have thus far not been successful.
Endotoxin extracts of the type used as a starting material to produce the RDE used in the present invention may be obtained from any Enterobacteriaciae including parent organisms and mutants. By way of example, the following genera are illustrative of the type of microorganisms that may be used:
Salmonella, Shigella, Escherichia, Brucella, Bordetella, Citrobacter, Pseudomonas, Pasturella, Neisseria, Proteus, Klebsiella, and Serratia.
The following species are typically employed:
S. minnesota, S. typhimuium, B. pertussis, B. abortus, S.enteritidis, E. coli, S. typhi, S. marcescens, S. typhosa, Shigella flexni, and S. abortus equi.
The endotoxic extracts used as a starting material may be prepared by one of several known methods [see, for example, Webster, M. E., Sagin, J. F., Landy, M., and Johnson, A. G., J. Immunol. 1955, 744, 55; Westphal, O., Luderitz, O., and Bister, F., Z. Naturforsch, 76 148 (1952); Westphal. O., Pyrogens, Polysaccharides in Biology, Tr. Second Macy Conference (George F. Springer, ed.), Madison, N.J. Madison Printing Co., 1957, 115; Galanos, C., Luderitz, O., Westphal, O., Eur. J. Biochem. 9, 245 (1969); Chen, C. H., Johnson, A. G., Kasai, N., Key, B. A., Levin, J., Nowotny, A., J. Infect. Dis. 128 543 (1973); Ribi, E., Haskins, W.T., Landy, M., Milner, K. C., The Journal of Experimental Medicine 114 647 (1961); Leive, L., Biochem. Biophys. Res. Comm. 21 290 (1965); and Ribi, E., Milner, K. C., and Perrine, T., J. Immunol. 82 75 (1959)].
A most suitable method of obtaining the endotoxic extract is that disclosed by Chen, et al.; namely, methanol-chloroform precipitation.
The methanol-chloroform precipitate (MCP) is reacted with an organic or inorganic acid and then lyophilized to produce a hydrolyzed crude lipid A with reduced toxicity and pyrogenicity as compared with the starting endotoxin material. The resulting product is then treated with a solvent which is capable of specifically dissolving fatty acids and other impurities without dissolving the crude lipid A. A suitable solvent for this purpose is acetone. The phosphate content of the detoxified, refined lipid A is about one-half that observed for the toxic counterpart suggesting that the phosphate content is related to the toxic effects of endotoxins.
Suitable inorganic acids used to react with MCP are hydrochloric acid, sulfuric acid or phosphoric acid and the suitable organic acids are toluene sulfonic acid or trichloroacetic acid. The reaction may be suitably conducted at a temperature between about 90.degree. and 130.degree. C. for a time sufficient to complete hydrolysis usually between about 15 and 60 minutes.
The preparation of crude detoxified endotoxin may also be accomplished by reacting the starting material with the selected acid in the presence of an organic solvent such as chloroform, methanol, and ethanol or combinations thereof.
The resulting crude lipid A is dissolved in acetone which is particularly suited to remove the fatty acid components. The solvent is then removed to produce crude detoxified endotoxin.
The crude detoxified endotoxin is then dissolved in a solvent and passed through a suitable chromatographic column such as, for example, a molecular exclusion chromatographic column, to separate the RDE fractions which are then combined after removal of the solvent. In one embodiment, the crude detoxified endotoxin solution is passed through a Sephadex column in the presence of a solvent such as chloroform, methanol, acetone, pyridine, ether or acetic acid or combinations thereof. The pressure of the column may vary but is typically in the range of between about atmospheric and 100 lbs/in.sup.2 and the flow rate is between about 0.1 and 10 ml/min.
Alternatively, the crude detoxified endotoxin solution is passed through a DEAE-cellulose column under the same pressure conditions as mentioned above for the Sephadex column. The flow rate may be maintained between about 2 and 15 ml/min. The solvents used are also the same as used for the Sephadex column although water and/or diethylamine can be added to all mixtures at a concentration of up to about 1%.
Other methods of producing RDE from crude detoxified endotoxin include passing the solution through a low pressure silica-gel 60 column having a particle size of between about 15 and 63 microns and using a suitable solvent such as chloroform, methanol, water or ammonium hydroxide. The preferred volume ratio of the aforementioned solvent mixture is about 50:25:4:2.
It is, therefore, an object of the present invention to employ a refined detoxified endotoxin product which can be effectively used to stimulate the immune system of a warm blooded animal. Specifically, RDE can be used as a B-cell mitogen, to stimulate the production of lymphokines, stimulate macrophages, and as an adjuvant which enhances the immune response of a warm blooded animal.